The present invention relates to the field of plant molecular biology, more particularly to high efficiency genetic manipulation and strong gene expression systems in species in the Pucciniomycotina and Ustilaginomycotina subphyla.
The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference, and for convenience are referenced in the following text by author and date and are listed alphabetically by author in the appended bibliography.
The Pucciniomycotina are a subphylum of fungi in the phylum of Basidiomycota (Kirk et al., 2008). It holds many species that have important industrial applications. For example, a number of species in the Rhodosporidium and Sporidiobolus genera, such as Rhodosporidium toruloides (also known as Rhodotorula gracilis, Rhodosporidium glutinis, Rhodotorula glutinis, Torula koishikawensis and Torula rubescens) and Sporobolomyces salmonicolor, are oil-rich single-cell yeasts capable of high density fermentation (Hu et al., 2009; Meng et al., 2009). These species hold great potential as a host for the production of long chain hydrocarbons, such as triacylglycerol (TAG, or fat), fatty acid esters (biodiesel), fatty alcohols, alcohols, lactones, terpenoids and vitamins (Wu et al., 2010a; Wu et al., 2010b; Zhao et al., 2010a; Zhao et al., 2010b). Although a method based on the PEG-mediated transfection of protoplast have been reported in Rhodosporidium toruloides (Tully and Gilbert, 1985), the method is highly unreliable and requires an auxotrophic mutant for transformation. The transformation vector cannot be applied to genetic manipulation of industrials strains due the instability of the plasmid pHG2, which contains the Phenylalanine Ammonia-lyase (PAL)-coding gene (PAL) of R. toruloides and LEU2 gene of Saccharomyces cerevisiae as a selection marker, and the site-specific pattern of DNA vector integration. Similarly, there are no functional promoters that can be used to drive the expression of utility genes or selections markers in Rhodosporidium. Similar situation is found in Sporobolomyces (Ianiri et al., 2011). In another example, species in Ustilaginomycotina subphylum, in particular, Ustilago and Pseudozyma genera, are known to produce glycolipids, which may function as a surfactant or fungicide (Hewald et al., 2005; Teichmann et al., 2010).
A complete genetic manipulation and expression system is typically composed of promoters that are either constitutive or inducible; selection markers; DNA vectors; methods to introduce the DNA into the host cell, either to integrate into the genome or replicate as an episome; and methods to inhibit or block expression of genes of interest.
Agrobacterium tumefaciens-mediated transformation (ATMT) is a convenient method for transformation of many fungal species (De Groot et al., 1998). Transformation efficiency may be improved by optimization of pH value for the Agrobacterium, ratio and absolute value of recipient cells and donor cells during co-culture (Ji et al., 2010) and the use of enhancer DNA sequences derived from T-DNA (YE and Gilbertson, 2009). In ATMT of plant species several techniques have been reported to improve transformation, including sonication and vacuum infiltration of plant tissues (de Oliveira et al., 2009); stronger promoters to drive expression of selection markers (Maehara et al., 2010) and control of host defense response (Khanna et al., 2007; Vega et al., 2008). The effects of these modifications have not been confirmed in the ATMT of fungi.
It is well-known that fungal cells may also be transformed by electroporation of either intact cells or protoplast (Wu and Letchworth, 2004); and transfection of a protoplast (Meyer, 2008; Turgeon et al., 2010) or simple chemical induction to increase cell wall permeability (Gietz and Woods, 2002; Hill et al., 1991; Ito et al., 1983). Random insertional mutagenesis is a powerful tool for fast identification of unknown genes. Although restriction enzyme-mediated integration (REMI) may be used to improve integration of linearized DNA vectors in a PEG-mediated transformation protocol (Bölker et al., 1995; Maier and Schafer, 1999), this method is hampered by large deletions of genomic DNA, multiple insertions and untagged mutagenesis including chromosomal rearrangements (Bölker et al., 1995; Meyer et al., 2003; Sweigard et al., 1998). On the other hand, ATMT has been recognized as a superior tool on this aspect (Choi et al., 2007; Soltani et al., 2008).
Selection of fungal transformants has been demonstrated with artificial constructs that express a protein that modifies the antibiotic or herbicide. Commonly used genes include hygromycin phosphotransferase (hpt) that confers resistance to Hygromycin B (Bundock et al., 1995); nourseothricin acetyltransferase (nat) that confers resistance to Nourseothricin (Ji et al., 2010; Krugel et al., 1988), aminoglycoside 3′-phosphotransferase (aph) or Neomycin phosphotransferase (npt) that confers resistance to Kanamycin or G418 or Neomycin (Goldstein and McCusker, 1999; Scorer et al., 1994), Streptoalloteichus hindustanus bleomycin gene (ble) that confers resistance to Zeocin (Pfeifer et al., 1997; Takeno et al., 2005); 5-enolpyruvyl-3-phosphoshikimate synthetase (aroA) gene confers resistance to the herbicide Glyphosate (Comai et al., 1983); phosphinothricin acetyl transferase (pat) that confers resistance to the herbicide bialaphos (Goldstein and McCusker, 1999); acetolactate synthase (acs) gene that confers resistance to the herbicide Sulfonylureas (Haughn et al., 1988).
Gene deletion and replacement are vital gene-targeting techniques in modern genetics. However, it is often very challenging to generate such mutants due to the low gene-targeting frequency. Techniques that significantly improve gene-targeting frequency are highly sought after in many organisms.
In higher eukaryotic DNA nonhomologous end joining (NHEJ) system, the DNA-dependent protein kinase (DNA-PK) holoenzyme comprises a polypeptide heterodimer of approximately 70 and 80 kDa, known as Ku70 and Ku80, which binds to DNA strand breaks, thereby recruiting and activating the 470-kDa catalytic subunit, termed as DNA-PKcs (Smith and Jackson, 1999). Whilst Rad51 and Rad52 are essential for the repair of DSB in the HR pathway (van Attikum et al., 2003), DNA-PKcs/Ku complex and XRCC4/ligase IV are vital in the NHEJ pathway in mammalian systems (van Attikum et al., 2001). However, homolog for the DNA-PKcs subunit remains unidentified in fungi. In recent years, there have been several reports of success on improvement of gene deletion frequency through disruption of the NHEJ pathway by deleting one or more of its key components (Kück and Hoff, 2010). This technique is cumbersome to apply.
On the other hand, a large number of compounds have been reported to inhibit the activity of DNA-PK, including wortmanin (Boulton et al., 1996), LY294002 (Rosenzweig et al., 1997), vanillin (Durant and Karran, 2003), NU1025 (Boulton et al., 1999), PD128763 (Tentori et al., 2002), AG14361 (Skalitzky et al., 2003), NU7026 [2-(morpholin-4-yl)-benzo[h]chomen-4-one; 2-(4-morpholinyl)-4H-naphthol[1,2-b]pyran-4-one] and NU7441 [8-(4-dibenzothienyl)-2-(4-morpholinyl)-4H-1-benzopyran-4-one]. The latter two are believed to be more specific and potent inhibitors of DNA-PK in animals (Veuger et al., 2003; Willmore et al., 2004). In the absence of DNA-PK in fungi, it is not known if there compounds will facilitate gene targeting.
Currently, genetic transformation of species in the Rhodosporidium, Sporobolomyces, Sporisorium and Ustilago genera is either completely not available or inefficient, and is a major hurdle to the advancement of renewable chemicals and biofuels.